Abstract. Traditional tests for developmental neurotoxicity have involved animal studies with behavioral or anatomical readouts that are integrative of toxicant effects on the whole organism. However, they are not informative of mechanism of action, particularly with molecular detail. Recently, methods to produce 3D brain organoids from stem cells were developed. These cortical organoids exhibit much more of the spatial and cell type organization of authentic cortical regions of the developing brain. We propose to develop a novel genetic screen of neurotoxicant effects on brain organoid development. The proposed project brings together several established as well as recent emerging technologies with a novel panel of over 1,000 genetically diverse ES cell lines to solve a problem that has been a barrier to developmental neurotoxicity (DNT) testing. Bulk harvesting of RNA required that expression studies use uniform cell populations. However, neurodevelopment is an inherently cell heterogenous process. In this project, we will use brain organoids produced in vitro from panel of genetically diverse ES cell lines in combination with single cell RNAseq to develop a scalable, highly efficient and highly granular eQTL DNT screen. The key innovations of our technology is (1) combining complex trait genetics with single cell transcriptomes, and (2) defining subpopulations of differentiating neural cell types using single cell transcriptomes, then assigning individual genetically diverse cells to the appropriate subpopulation. In this way, the muddle of data from heterogenous cell mixtures is avoided.!